Heme is a chemically reactive biological form of iron that may be toxic and pathogenic in certain clinical conditions, such as acute and chronic hemolysis, stroke, cerebral hemorrhage, sickle cell disease (SCD), shock and sepsis. Heme is also released by mechanical lysis of red blood cells during prolonged extracorporeal circulation for cardiac surgery or when heart valves malfunction. Hemopexin (HPX) is a heme binding protein that protects cells against these toxic effects of heme. The name hemopexin means heme fixer or grabber and when this protein binds heme in a 1:1 stoichiometric complex, we term it heme-HPX. Thus, as used herein, the terms HPX and apo-HPX refer to the protein, hemopexin, itself (not “pexin”). This nomenclature differs from that used for hemoglobin, which refers to heme bound to the protein, globin. Significantly, HPX can be inactivated, or lose its heme-binding ability, after exposure to conditions that generate reactive nitrogen species (RNS), which nitrate certain amino acids residues, e.g. tyrosine. Nitration of HPX reduces the levels of active HPX in the blood plasma and other biological fluids (e.g. cerebrospinal fluid, CSF) and allows heme toxicity to develop. Evidence supports that chemically-modified hemopexin, which has the heme coordinating histidines inactivated, remains in the circulation, and presumably also in other biological fluids like CSF, and is not rapidly cleared.
Many proteins are inactivated by oxidative modification(s), and we considered that the heme binding by hemopexin might become impaired in inflammation when reactive oxygen species (ROS) including hypochlorous acid (HOCl) are present. The potential for damage to hemopexin was revealed by our recent in vitro studies showing that the heme-hemopexin complex was highly resistant to oxidative damage from peroxides and HOCl compared with the apo-protein but high amounts of ROS in vitro were required. We investigated whether there is damage to HPX from RNS because in inflammatory conditions both ROS and RNS are present. For example, activated neutrophils produce their defensive “respiratory burst” of reactive oxygen intermediates; and endothelial cells, macrophages and astrocytes produce nitric oxide (NO.) after immunological or inflammatory stimuli. We have discovered that human (Homo sapiens), rabbit (Oryctolagus cuniculus), and rat (Rattus norvegicus) HPX isolated from plasma using standard techniques contain low levels of endogenous nitrated tyrosines. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified a predominant nitrated tyrosine that resides in the peptide YYnCFQGNQFLR (SEQ ID NO 1), and which is conserved in human, rabbit and rat HPX. Immuno-blotting and selective reaction monitoring were used to quantitate the extent of nitration of HPX after myeloperoxidase/glucose oxidase (MPO/GO) treatment an in vitro system that mimics oxidative stress with RNS. Significantly, heme binding by HPX is increasingly impaired as tyrosine nitration proceeds and two of the three nitrated tyrosines identified by our analysis reside in the heme binding site and interact directly with the heme. Importantly, nitration is an event requiring a specific environment around the tyrosine residue and lack of steric hindrance since this amino acid is often embedded within the hydrophobic core of the protein and protected from nitration. Thus, nitration of tyrosine residues is selective and such specificity strongly supports the concept that these amino acids are a preferential target for biological events in vivo.
Oxidative stress is linked to the development of neurodegeneration in the brain and inflammation is considered to contribute to the early stages of Alzheimer's disease (AD) to which HPX has been linked. Furthermore, brain iron increases in AD and in HPX-knockout mice as they age. We have previously shown that three models of ROS, namely H2O2, tert-butyl hydroperoxide and hypochlorous acid can impair heme binding by HPX, although high molar ratios of ROS:HPX are needed, generally supra-physiological. Here, we identify covalent oxidative modifications (COMs) of apo-HPX after exposure to these ROS. The term COM is used rather than post-translational modifications because COMs affect the mature, secreted HPX rather than the post-translational modifications that occur during the synthesis and secretion of HPX. Several tyrosine residues that reside in the heme binding site were targeted for modification by more than one oxidant and, thus, constitute susceptible targets for damage in vivo. In contrast, the heme-HPX complex is fairly resistant to damage by ROS, as previously shown and is shown here to be relatively resistant to nitration by MPO/GO. Even when heme-HPX is exposed to H2O2 or tert-butyl hydroperoxide, it still delivers heme for heme oxygenase-1 induction that is cytoprotective. Inactivation of apo-HPX in plasma or other biological fluids by oxidative modification likely occurs close to activated endothelial and immune system cells, which are the sites of production of RNS and ROS.
It has been shown that HPX loses this protective biological function when exposed to nitrating species in systems that mimic the oxidative environment of inflammation expected in sickle cell disease, shock and sepsis or in the brain after damage. Accordingly, HPX can be used as a biomarker for conditions of oxidative stress in which nitration of protein molecules has been detected including neurodegeneration (i.e., mild cognitive impairment (MCI) and Alzheimer's disease (AD)); in systemic inflammatory responses (i.e., acute lung injury, SCD, sepsis, shock and multiple organ failure syndrome); and is also of importance for neonates and children with hemolytic conditions and sepsis who are more at risk than adults due to their low basal levels of HPX. Further, two independent studies have shown that low levels of HPX in humans correlate with high risk of dying from sepsis.
It has been demonstrated that heme toxicity due to HPX depletion in mouse models of SCD and sepsis can be rescued by HPX supplementation. Thus, treatment with HPX infusions can aid in recovery and improve clinical outcomes in clinical SCD crisis and sepsis among other conditions. Nitration of HPX, which inactivates HPX's heme-binding ability, likely precedes its depletion because photo-inactivated HPX with altered heme coordinating histidine residues remains in the circulation. Low levels of HPX in humans when diagnosed with sepsis correlates with a high risk of dying and HPX supplementation in a mouse model of sepsis decreases the death rate from 80 to 20%. Plasma protein therapy is established for human serum albumin as a plasma expander to stabilize blood pressure in shock or sepsis and for immunoglobulins in certain immuno-deficiency states. HPX replenishment therapy is under consideration for commercial development. Current techniques measure solely total levels of HPX, which can underestimate the amount of active HPX. Therefore, there is a need for a sensitive and accurate assay for detecting the levels of nitrated HPX in samples of body fluid such as serum, plasma, cerebral spinal fluid and lung aspirate. Such information together with total HPX levels will allow a better understanding of the patients' risk of developing HPX deficiency and aid in diagnosis as well as in the timing of replenishment infusions.